Synthetic Chromosome Rearrangement And Modification By loxP-mediated Evolution Research Articles

Large-scale genomic rearrangements boost SCRaMbLE in Saccharomyces cerevisiae

Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) is a promising tool to study genomic rearrangements. However, the potential of SCRaMbLE to study genomic rearrangements is currently hindered, because a strain containing all 16 synthetic chromosomes is not yet available. Here, we construct SparLox83R, a yeast strain containing 83 loxPsym sites distributed across all 16 chromosomes. SCRaMbLE of SparLox83R produces versatile genome-wide genomic rearrangements, including inter-chromosomal events. Moreover, when combined with synthetic chromosomes, SCRaMbLE of hetero-diploids with SparLox83R leads to increased diversity of genomic rearrangements and relatively faster evolution of traits compared to hetero-diploids only with wild-type chromosomes. Analysis of the SCRaMbLEd strain with increased tolerance to nocodazole demonstrates that genomic rearrangements can perturb the transcriptome and 3D genome structure and consequently impact phenotypes. In summary, a genome with sparsely distributed loxPsym sites can serve as a powerful tool for studying the consequence of genomic rearrangements and accelerating strain engineering in Saccharomyces cerevisiae.

Dissecting aneuploidy phenotypes by constructing Sc2.0 chromosome VII and SCRaMbLEing synthetic disomic yeast

Aneuploidy compromises genomic stability, often leading to embryo inviability, and is frequently associated with tumorigenesis and aging. Different aneuploid chromosome stoichiometries lead to distinct transcriptomic and phenotypic changes, making it helpful to study aneuploidy in tightly controlled genetic backgrounds. By deploying the engineered SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution) system to the newly synthesized megabase Sc2.0 chromosome VII (synVII), we constructed a synthetic disomic yeast and screened hundreds of SCRaMbLEd derivatives with diverse chromosomal rearrangements. Phenotypic characterization and multi-omics analysis revealed that fitness defects associated with aneuploidy could be restored by (1) removing most of the chromosome content or (2) modifying specific regions in the duplicated chromosome. These findings indicate that both chromosome copy number and specific chromosomal regions contribute to the aneuploidy-related phenotypes, and the synthetic chromosome resource opens new paradigms in studying aneuploidy.

Dynamics of synthetic yeast chromosome evolution shaped by hierarchical chromatin organization.

Synthetic genome evolution provides a dynamic approach for systematically and straightforwardly exploring evolutionary processes. Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) is an evolutionary system intrinsic to the synthetic yeast genome that can rapidly drive structural variations. Here, we detect over 260 000 rearrangement events after the SCRaMbLEing of a yeast strain harboring 5.5 synthetic yeast chromosomes (synII, synIII, synV, circular synVI, synIXR and synX). Remarkably, we find that the rearrangement events exhibit a specific landscape of frequency. We further reveal that the landscape is shaped by the combined effects of chromatin accessibility and spatial contact probability. The rearrangements tend to occur in 3D spatially proximal and chromatin-accessible regions. The enormous numbers of rearrangements mediated by SCRaMbLE provide a driving force to potentiate directed genome evolution, and the investigation of the rearrangement landscape offers mechanistic insights into the dynamics of genome evolution.

Systematic dissection of key factors governing recombination outcomes by GCE-SCRaMbLE

With the completion of Sc2.0 chromosomes, synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) becomes more critical for in-depth investigation of fundamental biological questions and screening of industrially valuable characteristics. Further applications, however, are hindered due to the lack of facile and tight regulation of the SCRaMbLE process, and limited understanding of key factors that may affect the rearrangement outcomes. Here we propose an approach to precisely regulate SCRaMbLE recombination in a dose-dependent manner using genetic code expansion (GCE) technology with low basal activity. By systematically analyzing 1380 derived strains and six yeast pools subjected to GCE-SCRaMbLE, we find that Cre enzyme abundance, genome ploidy and chromosome conformation play key roles in recombination frequencies and determine the SCRaMbLE outcomes. With these insights, the GCE-SCRaMbLE system will serve as a powerful tool in the future exploitation and optimization of the Sc2.0-related technologies.

Directed yeast genome evolution by controlled introduction of trans-chromosomic structural variations.

Naturally occurring structural variations (SVs) are a considerable source of genomic variation that can reshape the 3D architecture of chromosomes. Controllable methods aimed at introducing the complex SVs and their related molecular mechanisms have remained farfetched. In this study, an SV-prone yeast strain was developed using Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) technology with two synthetic chromosomes, namely synV and synX. The biosynthesis of astaxanthin is used as a readout and a proof of concept for the application of SVs in industries. Our findings showed that complex SVs, including a pericentric inversion and a trans-chromosome translocation between synV and synX, resulted in two neo-chromosomes and a 2.7-fold yield of astaxanthin. Also, genetic targets were mapped, which resulted in a higher astaxanthin yield, thus demonstrating the SVs' ability to reorganize genetic information along the chromosomes. The rational design of trans-chromosome translocation and pericentric inversion enabled precise induction of these phenomena. Collectively, this study provides an effective tool to not only accelerate the directed genome evolution but also to reveal the mechanistic insight of complex SVs for altering phenotypes.

Genomic Iterative Replacements of Large Synthetic DNA Fragments in Corynebacterium glutamicum.

Synthetic genomics will advance our understanding of life and allow us to rebuild the genomes of industrial microorganisms for enhancing performances. Corynebacterium glutamicum, a Gram-positive bacterium, is an important industrial workhorse. However, its genome synthesis is impeded by the low efficiencies in DNA delivery and in genomic recombination/replacement. In the present study, we describe a genomic iterative replacement system based on RecET recombination for C. glutamicum, involving the successive integration of up to 10 kb DNA fragments obtained in vitro, and the transformants are selected by the alternative use of kanR and speR selectable markers. As a proof of concept, we systematically redesigned and replaced a 54.3 kb wild-type sequence of C. glutamicumATCC13032 with its 55.1 kb synthetic counterpart with several novel features, including decoupled genes, the standard PCRTags, and 20 loxPsym sites, which was for the first time incorporated into a bacterial genome. The resulting strain semi-synCG-A1 had a phenotype and fitness similar to the wild-type strain under various stress conditions. The stability of the synthetic genome region faithfully maintained over 100 generations of nonselective growth. Genomic deletions, inversions, and translocations occurred in the synthetic genome region upon induction of synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE), revealing potential genetic flexibility for C. glutamicum. This strategy can be used for the synthesis of a larger region of the genome and facilitate the endeavors for metabolic engineering and synthetic biology of C. glutamicum.

Systematical Engineering of Synthetic Yeast for Enhanced Production of Lycopene.

Synthetic biology allows the re-engineering of biological systems and promotes the development of bioengineering to a whole new level, showing great potential in biomanufacturing. Here, in order to make the heterologous lycopene biosynthesis pathway compatible with the host strain YSy 200, we evolved YSy200 using a unique Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) system that is built in the Sc2.0 synthetic yeast. By inducing SCRaMbLE, we successfully identified a host strain YSy201 that can be served as a suitable host to maintain the heterologous lycopene biosynthesis pathway. Then, we optimized the lycopene biosynthesis pathway and further integrated into the rDNA arrays of YSy201 to increase its copy number. In combination with culturing condition optimization, we successfully screened out the final yeast strain YSy222, which showed a 129.5-fold increase of lycopene yield in comparison with its parental strain. Our work shows that, the strategy of combining the engineering efforts on both the lycopene biosynthesis pathway and the host strain can improve the compatibility between the heterologous pathway and the host strain, which can further effectively increase the yield of the target product.

Compacting a synthetic yeast chromosome arm

BackgroundRedundancy is a common feature of genomes, presumably to ensure robust growth under different and changing conditions. Genome compaction, removing sequences nonessential for given conditions, provides a novel way to understand the core principles of life. The synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) system is a unique feature implanted in the synthetic yeast genome (Sc2.0), which is proposed as an effective tool for genome minimization. As the Sc2.0 project is nearing its completion, we have begun to explore the application of the SCRaMbLE system in genome compaction.ResultsWe develop a method termed SCRaMbLE-based genome compaction (SGC) and demonstrate that a synthetic chromosome arm (synXIIL) can be efficiently reduced. The pre-introduced episomal essential gene array significantly enhances the compacting ability of SGC, not only by enabling the deletion of nonessential genes located in essential gene containing loxPsym units but also by allowing more chromosomal sequences to be removed in a single SGC process. Further compaction is achieved through iterative SGC, revealing that at least 39 out of 65 nonessential genes in synXIIL can be removed collectively without affecting cell viability at 30 °C in rich medium. Approximately 40% of the synthetic sequence, encoding 28 genes, is found to be dispensable for cell growth at 30 °C in rich medium and several genes whose functions are needed under specified conditions are identified.ConclusionsWe develop iterative SGC with the aid of eArray as a generic yet effective tool to compact the synthetic yeast genome.

Chromosomal Rearrangements of Synthetic Yeast by SCRaMbLE.

Diversified genomes derived from chromosomal rearrangements are valuable materials for evolution. Naturally, chromosomal rearrangements occur at extremely low frequency to ensure genome stability. In the synthetic yeast genome project (Sc2.0), an inducible chromosome rearrangement system named Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) is built to produce chromosomal rearrangements such as deletion, duplication, inversion, and translocation at high efficiency. Here, we detail the method to activate SCRaMbLE in a synthetic strain, to analyze the SCRaMbLEd genome, and to dissect the causative rearrangements for a desired phenotype after SCRaMbLEing.

SCRaMbLE-in: A Fast and Efficient Method to Diversify and Improve the Yields of Heterologous Pathways in Synthetic Yeast.

The synthetic chromosome rearrangement and modification by LoxP-mediated evolution (SCRaMbLE) system is a key component of the synthetic yeast genome (Sc2.0) project, an international effort to construct an entire synthetic genome in yeast. SCRaMbLE involves the introduction of thousands of symmetrical LoxP (LoxPsym) recombination sites downstream of every nonessential gene in all 16 chromosomes, enabling numerous genome rearrangements in the form of deletions, inversions, duplications, and translocations by the Cre-LoxPsym recombination system. We highlight a two-step protocol for SCRaMbLE-in (Liu, Nat Commun 9(1):1936, 2018), a recombinase-based combinatorial method to expedite genetic engineering and exogenous pathway optimization, using a synthetic β-carotene pathway as an example. First, an in vitro phase uses a recombinase toolkit to diversify gene expression by integrating various regulatory elements into the target pathway. This combinatorial pathway library can be transformed directly into yeast for traditional screening. Once an optimized pathway which is flanked by LoxPsym sites is identified, it is transformed into Sc2.0 yeast for the in vivo SCRaMbLE phase, where LoxPsym sites in the synthetic yeast genome and Cre recombinase catalyze massive genome rearrangements. We describe all the conditions necessary to perform SCRaMbLE and post-SCRaMbLE experiments including screening, spot test analysis, and PCRTag analysis to elucidate genotype-phenotype relationships.

Improving prodeoxyviolacein production via multiplex SCRaMbLE iterative cycles

The synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) system has been used to improve prodeoxyviolacein (PDV) production in haploid yeast containing chromosome synV. To rapidly and continuously generate genome diversification with the desired phenotype, the multiplex SCRaMbLE iterative cycle strategy has been developed for the screening of high PDV production strains. Wholegenome sequencing analysis reveals large duplications, deletions, and even the whole genome duplications. The deletion of YER151C is proved to be responsible for the increase. This study demonstrates that artificial DNA rearrangement can be used to accelerate microbial evolution and the production of biobased chemicals.

Heterozygous diploid and interspecies SCRaMbLEing

SCRaMbLE (Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution) is a genome restructuring technique that can be used in synthetic genomes such as that of Sc2.0, the synthetic yeast genome, which contains hundreds to thousands of strategically positioned loxPsym sites. SCRaMbLE has been used to induce rearrangements in yeast strains harboring one or more synthetic chromosomes, as well as plasmid DNA in vitro and in vivo. Here we describe a collection of heterozygous diploid strains produced by mating haploid semisynthetic Sc2.0 strains to haploid native parental strains. We subsequently demonstrate that such heterozygous diploid strains are more robust to the effects of SCRaMbLE than haploid semisynthetic strains, rapidly improve rationally selected phenotypes in SCRaMbLEd heterozygous diploids, and establish that multiple sets of independent genomic rearrangements are able to lead to similar phenotype enhancements. Finally, we show that heterozygous diploid SCRaMbLE can also be carried out in interspecies hybrid strains.

Precise control of SCRaMbLE in synthetic haploid and diploid yeast

Compatibility between host cells and heterologous pathways is a challenge for constructing organisms with high productivity or gain of function. Designer yeast cells incorporating the Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) system provide a platform for generating genotype diversity. Here we construct a genetic AND gate to enable precise control of the SCRaMbLE method to generate synthetic haploid and diploid yeast with desired phenotypes. The yield of carotenoids is increased to 1.5-fold by SCRaMbLEing haploid strains and we determine that the deletion of YEL013W is responsible for the increase. Based on the SCRaMbLEing in diploid strains, we develop a strategy called Multiplex SCRaMbLE Iterative Cycling (MuSIC) to increase the production of carotenoids up to 38.8-fold through 5 iterative cycles of SCRaMbLE. This strategy is potentially a powerful tool for increasing the production of bio-based chemicals and for mining deep knowledge.

3D organization of synthetic and scrambled chromosomes.

Although the design of the synthetic yeast genome Sc2.0 is highly conservative with respect to gene content, the deletion of several classes of repeated sequences and the introduction of thousands of designer changes may affect genome organization and potentially alter cellular functions. We report here the Hi-C-determined three-dimensional (3D) conformations of Sc2.0 chromosomes. The absence of repeats leads to a smoother contact pattern and more precisely tractable chromosome conformations, and the large-scale genomic organization is globally unaffected by the presence of synthetic chromosome(s). Two exceptions are synIII, which lacks the silent mating-type cassettes, and synXII, specifically when the ribosomal DNA is moved to another chromosome. We also exploit the contact maps to detect rearrangements induced in SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution) strains.

Deep functional analysis of synII, a 770-kilobase synthetic yeast chromosome

Here, we report the successful design, construction, and characterization of a 770-kilobase synthetic yeast chromosome II (synII). Our study incorporates characterization at multiple levels-including phenomics, transcriptomics, proteomics, chromosome segregation, and replication analysis-to provide a thorough and comprehensive analysis of a synthetic chromosome. Our Trans-Omics analyses reveal a modest but potentially relevant pervasive up-regulation of translational machinery observed in synII, mainly caused by the deletion of 13 transfer RNAs. By both complementation assays and SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution), we targeted and debugged the origin of a growth defect at 37°C in glycerol medium, which is related to misregulation of the high-osmolarity glycerol response. Despite the subtle differences, the synII strain shows highly consistent biological processes comparable to the native strain.

SCRaMbLE generates designed combinatorial stochastic diversity in synthetic chromosomes.

Synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) generates combinatorial genomic diversity through rearrangements at designed recombinase sites. We applied SCRaMbLE to yeast synthetic chromosome arm synIXR (43 recombinase sites) and then used a computational pipeline to infer or unscramble the sequence of recombinations that created the observed genomes. Deep sequencing of 64 synIXR SCRaMbLE strains revealed 156 deletions, 89 inversions, 94 duplications, and 55 additional complex rearrangements; several duplications are consistent with a double rolling circle mechanism. Every SCRaMbLE strain was unique, validating the capability of SCRaMbLE to explore a diverse space of genomes. Rearrangements occurred exclusively at designed loxPsym sites, with no significant evidence for ectopic rearrangements or mutations involving synthetic regions, the 99% nonsynthetic nuclear genome, or the mitochondrial genome. Deletion frequencies identified genes required for viability or fast growth. Replacement of 3′ UTR by non-UTR sequence had surprisingly little effect on fitness. SCRaMbLE generates genome diversity in designated regions, reveals fitness constraints, and should scale to simultaneous evolution of multiple synthetic chromosomes.

Synthetic genome technology for yeast

The authors have constructed a synthetic chromosome arm of the yeast Saccharomyces cerevisiae, laying the groundwork for creating a synthetic eukaryotic genome. A ~90 kb stretch of the right arm of chromosome IX was designed in silico to remove unstable elements such as transposons and to facilitate genome manipulations. The synthetic sequence, which was swapped into the endogenous genomic region, also contains an inducible evolution system, SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution), which triggers combinatorial rearrangements to allow the study of complex phenotypes.

Synthetic chromosome arms function in yeast and generate phenotypic diversity by design

Recent advances in DNA synthesis technology have enabled the construction of novel genetic pathways and genomic elements, furthering our understanding of system-level phenomena. The ability to synthesize large segments of DNA allows the engineering of pathways and genomes according to arbitrary sets of design principles. Here we describe a synthetic yeast genome project, Sc2.0, and the first partially synthetic eukaryotic chromosomes, Saccharomyces cerevisiae chromosome synIXR, and semi-synVIL. We defined three design principles for a synthetic genome as follows: first, it should result in a (near) wild-type phenotype and fitness; second, it should lack destabilizing elements such as tRNA genes or transposons; and third, it should have genetic flexibility to facilitate future studies. The synthetic genome features several systemic modifications complying with the design principles, including an inducible evolution system, SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution). We show the utility of SCRaMbLE as a novel method of combinatorial mutagenesis, capable of generating complex genotypes and a broad variety of phenotypes. When complete, the fully synthetic genome will allow massive restructuring of the yeast genome, and may open the door to a new type of combinatorial genetics based entirely on variations in gene content and copy number.